Fibril formation and ordering of disordered FUS LC driven by hydrophobic interactions.

Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure. Here we investigate the role of hydrophobic interactions by studying the low-complexity domain of the disordered 'fused in sarcoma' (FUS) protein at the air/water interface. Using surface-specific microscopic and spectroscopic techniques, we find that a hydrophobic interface drives fibril formation and molecular ordering of FUS, resulting in solid-like film formation. This phase transition occurs at 600-fold lower FUS concentration than required for the canonical FUS low-complexity liquid droplet formation in bulk. These observations highlight the importance of hydrophobic effects for protein phase separation and suggest that interfacial properties drive distinct protein phase-separated structures.

Lipid-driven condensation and interfacial ordering of FUS.

Protein condensation into liquid-like structures is critical for cellular compartmentalization, RNA processing, and stress response. Research on protein condensation has primarily focused on membraneless organelles in the absence of lipids. However, the cellular cytoplasm is full of lipid interfaces, yet comparatively little is known about how lipids affect protein condensation. Here, we show that nonspecific interactions between lipids and the disordered fused in sarcoma low-complexity (FUS LC) domain strongly affect protein condensation. In the presence of anionic lipids, FUS LC formed lipid-protein clusters at concentrations more than 30-fold lower than required for pure FUS LC. Lipid-triggered FUS LC clusters showed less dynamic protein organization than canonical, lipid-free FUS LC condensates. Lastly, we found that phosphatidylserine membranes promoted FUS LC condensates having ß sheet structures, while phosphatidylglycerol membranes initiated unstructured condensates. Our results show that lipids strongly influence FUS LC condensation, suggesting that protein-lipid interactions modulate condensate formation in cells.

Phospholipid acyl tail affects lipid headgroup orientation and membrane hydration.

Biomembrane hydration is crucial for understanding processes at biological interfaces. While the effect of the lipid headgroup has been studied extensively, the effect (if any) of the acyl chain chemical structure on lipid-bound interfacial water has remained elusive. We study model membranes composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids, the most abundant lipids in biomembranes. We explore the extent to which the lipid headgroup packing and associated water organization are affected by the lipid acyl tail unsaturation and chain length. To this end, we employ a combination of surface-sensitive techniques, including sum-frequency generation spectroscopy, surface pressure measurements, and Brewster angle microscopy imaging. Our results reveal that the acyl tail structure critically affects the headgroup phosphate orientational distribution and lipid-associated water molecules, for both PE and PC lipid monolayers at the air/water interface. These insights reveal the importance of acyl chain chemistry in determining not only membrane fluidity but also membrane hydration.

In Situ Label-Free Study of Protein Adsorption on Nanoparticles.

Improving the design of nanoparticles for use as drug carriers or biosensors requires a better understanding of the protein-nanoparticle interaction. Here, we present a new tool to investigate this interaction in situ and without additional labeling of the proteins and/or nanoparticles. By combining nonresonant second-harmonic light scattering with a modified Langmuir model, we show that it is possible to gain insight into the adsorption behavior of blood proteins, namely fibrinogen, human serum albumin, and transferrin, onto negatively charged polystyrene nanoparticles. The modified Langmuir model gives us access to the maximum amount of adsorbed protein, the apparent binding constant, and Gibbs free energy. Furthermore, we employ the method to investigate the influence of the nanoparticle size on the adsorption of human serum albumin and find that the amount of adsorbed protein increases more than the surface area per nanoparticle for larger diameters.

Water at charged interfaces.

The ubiquity of aqueous solutions in contact with charged surfaces and the realization that the molecular-level details of water-surface interactions often determine interfacial functions and properties relevant in many natural processes have led to intensive research. Even so, many open questions remain regarding the molecular picture of the interfacial organization and preferential alignment of water molecules, as well as the structure of water molecules and ion distributions at different charged interfaces. While water, solutes and charge are present in each of these systems, the substrate can range from living tissues to metals. This diversity in substrates has led to different communities considering each of these types of aqueous interface. In this Review, by considering water in contact with metals, oxides and biomembranes, we show the essential similarity of these disparate systems. While in each case the classical mean-field theories can explain many macroscopic and mesoscopic observations, it soon becomes apparent that such theories fail to explain phenomena for which molecular properties are relevant, such as interfacial chemical conversion. We highlight the current knowledge and limitations in our understanding and end with a view towards future opportunities in the field.

Location and Conformation of the LKα14 Peptide in Water/Ethanol Mixtures.

It is widely recognized that solvation is one of the major factors determining structure and functionality of proteins and long peptides, however it is a formidable challenge to address it both experimentally and computationally. For this reason, simple peptides are used to study fundamental aspects of solvation. It is well established that alcohols can change the peptide conformation and tuning of the alcohol content in solution can dramatically affect folding and, as a consequence, the function of the peptide. In this work, we focus on the leucine and lysine based LKα14 peptide designed to adopt an α-helical conformation at an apolar-polar interface. We investigate LKα14 peptide's bulk and interfacial behavior in water/ethanol mixtures combining a suite of experimental techniques (namely, circular dichroism and nuclear magnetic resonance spectroscopy for the bulk solution, surface pressure measurements and vibrational sum frequency generation spectroscopy for the air-solution interface) with molecular dynamics simulations. We observe that ethanol highly affects both the peptide location and conformation. At low ethanol content LKα14 lacks a clear secondary structure in bulk and shows a clear preference to reside at the air-solution interface. When the ethanol content in solution increases, the peptide's interfacial affinity is markedly reduced and the peptide approaches a stable α-helical conformation in bulk facilitated by the amphiphilic nature of the ethanol molecules.

Both Poly(ethylene glycol) and Poly(methyl ethylene phosphate) Guide Oriented Adsorption of Specific Proteins.

Developing new functional biomaterials requires the ability to simultaneously repel unwanted and guide wanted protein adsorption. Here, we systematically interrogate the factors determining the protein adsorption by comparing the behaviors of different polymeric surfaces, poly(ethylene glycol) and a poly(phosphoester), and five different natural proteins. Interestingly we observe that, at densities comparable to those used in nanocarrier functionalization, the same proteins are either adsorbed (fibrinogen, human serum albumin, and transferrin) or repelled (immunoglobulin G and lysozyme) by both polymers. However, when adsorption takes place, the specific surface dictates the amount and orientation of each protein.

Interfacial Conformation of Hydrophilic Polyphosphoesters Affects Blood Protein Adsorption.

Synthetic polymers are commonly used as protein repelling materials for a variety of biomedical applications. Despite their widespread use, the fundamental mechanism underlying protein repellence is often elusive. Such insights are essential for improving existing and developing new materials. Here, we investigate how subtle differences in the chemistry of hydrophilic polyphosphoesters influence the adsorption of the human blood proteins serum albumin and fibrinogen. Using thermodynamic measurements, surface-specific vibrational spectroscopy, and Brewster angle microscopy, we investigate protein adsorption, hydration, and steric repulsion properties of the polyphosphoester polymers. Whereas both surface hydration and polymer conformation of the polymers vary substantially as a consequence of the chemical differences in the polymer structure, the protein repellency ability of these hydrophilic materials appears to be dominated by steric repulsion.

Engineering Proteins at Interfaces: From Complementary Characterization to Material Surfaces with Designed Functions.

Once materials come into contact with a biological fluid containing proteins, proteins are generally-whether desired or not-attracted by the material's surface and adsorb onto it. The aim of this Review is to give an overview of the most commonly used characterization methods employed to gain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insight ideally paves the way for engineering functional materials that interact with proteins in a predetermined manner.

Saturation of charge-induced water alignment at model membrane surfaces.

The electrical charge of biological membranes and thus the resulting alignment of water molecules in response to this charge are important factors affecting membrane rigidity, transport, and reactivity. We tune the surface charge density by varying lipid composition and investigate the charge-induced alignment of water molecules using surface-specific vibrational spectroscopy and molecular dynamics simulations. At low charge densities, the alignment of water increases proportionally to the charge. However, already at moderate, physiologically relevant charge densities, water alignment starts to saturate despite the increase in the nominal surface charge. The saturation occurs in both the Stern layer, directly at the surface, and in the diffuse layer, yet for distinctly different reasons. Our results show that the soft nature of the lipid interface allows for a marked reduction of the surface potential at high surface charge density via both interfacial molecular rearrangement and permeation of monovalent ions into the interface.

Repelling and ordering: the influence of poly(ethylene glycol) on protein adsorption.

Development of new materials for drug delivery and biosensing requires the fine-tuning of interfacial properties. We report here the influence of the poly(ethylene glycol) (PEG) grafting density in model phospholipid monolayers on the adsorption behavior of bovine serum albumin and human fibrinogen, not only with respect to the amount of adsorbed protein, but also its orientational ordering on the surface. As expected, with increasing interfacial PEG density, the amount of adsorbed protein decreases up to the point where complete protein repellency is reached. However, at intermediate concentrations, the net orientation of adsorbed fibrinogen is highest. The different proteins respond differently to PEG, not only in the amount of protein adsorbed, but also in the manner that proteins adsorb. The results show that for specific cases, tuning the interfacial PEG concentration allows to guide the protein adsorption configuration, a feature sought after in materials for both biosensing and biomedical applications.

Surface-specific vibrational spectroscopy of the water/silica interface: screening and interference.

Surface-specific vibrational sum-frequency generation spectroscopy (V-SFG) is frequently used to obtain information about the molecular structure at charged interfaces. Here, we provide experimental evidence that not only screening of surface charges but also interference limits the extent to which V-SFG probes interfacial water at sub-mM salt concentrations. As a consequence, V-SFG yields information about the ∼single monolayer interfacial region not only at very high ionic strength, where the surface charge is effectively screened, but also for pure water due to the particularly large screening length at this low ionic strength. At these low ionic strengths, the large screening lengths cause destructive interference between contributions in the surface region. A recently proposed theoretical framework near-quantitatively describes our experimental findings by considering only interference and screening. However, a comparison between NaCl and LiCl reveals ion specific effects in the screening efficiency of different electrolytes. Independent of electrolyte, the hydrogen bonding strength of water right at the interface is enhanced at high electrolyte concentrations.

Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

Pushing the high-energy limit of plasmonics.

The localized surface plasmon resonance of metal nanoparticles allows confining the eletromagnetic field in nanosized volumes, creating high-field "hot spots", most useful for enhanced nonlinear optical spectroscopies. The commonly employed metals, Au and Ag, yield plasmon resonances only spanning the visible/near-infrared range. Stretching upward, the useful energy range of plasmonics requires exploiting different materials. Deep-ultraviolet plasmon resonances happen to be achievable with one of the cheapest and most abundant materials available: aluminum indeed holds the promise of a broadly tunable plasmonic response, theoretically extending far into the deep-ultraviolet. Complex nanofabrication and the unavoidable Al oxidation have so far prevented the achievement of this ultimate high-energy response. A nanofabrication technique producing purely metallic Al nanoparticles has at last allowed to overcome these limits, pushing the plasmon resonance to 6.8 eV photon energy (≈180 nm) and thus significantly broadening the spectral range of plasmonics' numerous applications.

Second harmonic light scattering from the surface of colloidal objects: theory and applications.

Because properties of colloids containing micro- and nano-particles are much influenced by the structure of, and interactions occurring at, the particle surface, it is important to be able to characterize the particle surface in situ and nondestructively. Nonlinear light scattering from colloidal particles has been developed into a powerful and versatile technique for characterizing particle surfaces since the first demonstration of the detection of second harmonic generation from molecules adsorbed on micrometer-sized colloidal particles by Eisenthal and co-workers (Wang, H.; Yan, E. C. Y.; Borguet, E.; Eisenthal, K. B. Second Harmonic Generation from the Surface of Centrosymmetric Particles in Bulk Solution. Chem. Phys. Lett. 1996, 259, 15-20). At present, second harmonic light scattering from the particle surface can be quantitatively described by theoretical models and used to measure the adsorption kinetics, molecular structure, and reaction rates at the surfaces of a variety of micrometer- to nanometer-sized particles, including biological cells.

Deep ultraviolet plasmon resonance in aluminum nanoparticle arrays.

Small aluminum nanoparticles have the potential to exhibit localized surface plasmon resonances in the deep ultraviolet region of the electromagnetic spectrum, however technical and scientific challenges make it difficult to attain this limit. We report the fabrication of arrays of Al/Al2O3 core/shell nanoparticles with a metallic-core diameter between 12 and 25 nm that display sharp plasmonic resonances at very high energies, up to 5.8 eV (down to λ = 215 nm). The arrays were fabricated by means of a straightforward self-organization approach. The experimental spectra were compared with theoretical calculations that allow the correlation of each feature to the corresponding plasmon modes.

Nonlinear light scattering and spectroscopy of particles and droplets in liquids.

Nano- and microparticles have optical, structural, and chemical properties that differ from both their building blocks and the bulk materials themselves. These different physical and chemical properties are induced by the high surface-to-volume ratio. As a logical consequence, to understand the properties of nano- and microparticles, it is of fundamental importance to characterize the particle surfaces and their interactions with the surrounding medium. Recent developments of nonlinear light scattering techniques have resulted in a deeper insight of the underlying light-matter interactions. They have shed new light on the molecular mechanism of surface kinetics in solution, properties of interfacial water in contact with hydrophilic and hydrophobic particles and droplets, molecular orientation distribution of molecules at particle surfaces in solution, interfacial structure of surfactants at droplet interfaces, acid-base chemistry on particles in solution, and vesicle structure and transport properties.

A self-assembled, metallo-organic supramolecular frequency doubler.

The dimeric self-assembly program of a new, multidentate ligand with cuprous ions overcomes crystal packing forces, which leads to C(i) symmetry in the solid state, to form a no less than partially C(2)-symmetric structure in solution. The resulting tetranitro-substituted dicopper(i) metallocyclophane displays an exceptionally strong second harmonic frequency response (ß = (3000 ± 600) × 10(-30) esu for a fundamental at 800 nm).

Communication: Reactions and adsorption at the surface of silver nanoparticles probed by second harmonic generation.

Even though nanoparticles have dimensions much smaller than the optical wavelength and shapes commonly with inversion symmetry, we show, for the first time, direct experimental evidence that second harmonic generation (SHG) can be detected from the surface layer of metallic nanoparticles, in this case 40 nm radius Ag particles. The SH intensity detected is shown to substantially decrease upon chemical bonding of thiol molecules to the Ag particle surface. The surface generated SH intensity can be used for probing properties and processes at the nanoparticle surface.